Kidney stones are a common & serious illness. Urinary citrate is the most important inhibitor of calcium stones by keeping calcium soluble. The regulation of citrate transport in the kidney has received inadequate investigation & remains poorly understood. A single transporter NaDC1 has been assumed to be responsible for intestinal & renal citrate reabsorption. However we have recently identified a novel calcium-sensitive citrate transport process. The overall objective of this application is to determine the mechanisms & regulation of kidney proximal tubule apical citrate transport via a novel apical calcium-sensitive transporter, & the relative roles of NaDC1 & NaDC3. Our central hypothesis is that the novel citrate reabsorptive process functions to limit citrate transport in the proximal tubule when calcium is increased. Elucidation of previously unidentified citrate transporters can potentially lead to new treatments to increase urinary citrate. Aims are: 1) Define the novel calcium-sensitive citrate transporter & its role in the proximal tubule. Our working hypothesis, based on data in three proximal tubule cell lines, is that in addition to NaDC1, a novel calcium- sensitive dicarboxylate transporter is present on the apical side of the proximal tubule. We will use knockdown of NaDC1 in these cell lines to demonstrate this. Also we will determine citrate transport rates & the extracellular calcium-sensitivity for the remaining citrate transport process found in proximal tubule cells grown from dissected proximal tubule segments from knockout mice. 2) Determine the role of NaDC3 in proximal tubule reabsorption of citrate. Based on our surprising preliminary data, we will determine if NaDC3 is expressed on the apical membrane & accounts for calcium- sensitive citrate reabsorption. We will determine the localization of NaDC3 in the proximal tubule & determine the calcium sensitivity on the apical surface. We will also determine the role of basolateral NaDC3. 3) Determine the role of NaDC1 in renal reabsorption & intestinal absorption of citrate. Our working hypothesis is that NaDC1 is not the exclusive mechanism of citrate transport in the kidney & intestine. Other apical mechanisms may both be important in conditions such as acidosis & hypercalciuria. We will use NaDC1 knockout mice & compare renal citrate reabsorption in the -/- to +/+ mice both under baseline & conditions of acidosis & hypercalciuria. We will also determine intestinal citrate absorption in NaDC1 knockout mice.. These studies will establish new paradigms in understanding citrate reabsorption in both the kidney proximal tubule & the intestine, & how these paradigms are impacted in acidosis & hypercalciuria. These outcomes are expected to have an important impact since understanding the regulation of citrate transport in the proximal tubule will dramatically improve strategies in the prevention of calcium stones. PUBLIC HEALTH RELEVANCE: Kidney stones are a common and serious medical disorder, causing significant medical costs. Urinary citrate is the most important inhibitor of calcium stones and low urinary citrate is a common contributor to many stone types. The proposed research is expected to identify new mechanisms of regulation of urinary citrate excretion. This contribution will be significant because it will transform our understanding of urinary citrate excretion and would be expected to lead to new treatment paradigms.